Radical-Containing PM0.2 Initiates Epithelial-to-Mesenchymal Transitions in Infant Airway

Epithelial Cells

Stephania A. Cormier, Ph.D. Department of Pharmacology & Experimental Therapeutics

Louisiana State University Health Sciences Center – New Orleans

Central Hypothesis

• Adult respiratory diseases result, in part, from environmental impact(s) that occur during a critical phase of pulmonary immuno-maturation. – Environment – Viral – Allergen

Infant

RSV

Adult Airways

Disease

Influenza PM

Allergen

The Infant Lung

• At birth - saccular

– Humans • 15% alveoli

– Rodents • 0% alveoli

• Postnatal development

– Humans • 3yr of age

– Rodents • 4-7d, alveoli

• 10d, respiratory bronchioles

*Pictures are artistic renditions of lung development and are designed to emphasize terminal acinus development and not the entire conducting airway system

Behrman: Nelson Textbook of Pediatrics, 16th ed., 2000. Langston C, et al. Am Rev Respir Dis. 1984;129:607-613.

PM INDUCES ADVERSE PULMONARY EFFECTS IN INFANTS

WHAT IN PM IS RESPONSIBLE FOR ADVERSE PULMONARY EVENTS?

Atmospheric Fine Particles Contain Persistent Semiquinone-type Radicals

PM2.5:1e16 - 1e17 radicals/g CS tar: 1e16 radicals/g

The existence of EPFRs represents a new paradigm for evaluating the toxicity of airborne PM2.5.

Combustion-Generated Fine Particles Contain Persistent Semiquinone-type Radicals

EPFRs in Baton Rouge PM2.5 PERSIST

T1/2 = 21d

Problems with Studying Atmospheric PM Samples

• Size variation

• Sample variation

• Chemical complexity

– Organic compounds

– Metal ions

• Sufficient sample size

• Collection methodology (e.g. CAPs, extracts, etc.)

Laboratory Generated Combustion Samples

• Control – Size – Chemical composition – Sufficient quantities

• In vivo inhalation studies

• More accurate assessment of potential risk posed by specific PM components – CHC/BHC – Radicals

Dellinger et al., 2007

Silica Silica Silica

CuO CuO

Silica

DCB/MCP

DCB/MCP

Days!

Particle Systems

EPFRs:1e14 - 1e14 radicals/g

UFP generated from the combustion processes increases oxidative stress resulting in pulmonary damage and/or changes in lung architecture ultimately resulting in adult airways disease (i.e., asthma).

Fetal Lung

Pulmonary Damage (epithelia, cilia, &/or

structure)

Chronic Inflammation Lung

(Asthma; COPD)

Changes in Pulmonary Immunity (Th2 Skew)

Chronic Inflammation Lung

(Asthma; COPD)

PM0.2 Hypothesis:

In Vivo Acute Exposure Protocol

Protocol Time (d)

Rat age (d)

Analysis

0 7 5

14 7 12

2

9

1

8

3

10

4

11

6

13

8

15

Study Endpoints Lung Function

AHR Resistance, elastance, compliance

Lung Histology Cellular inflammation Mucus production

Inflammation BAL: cell type & number, cytokine levels

Inhalation Exposure to EPFRs Induces Physiologic Response in Neonates

Balakrishna S, Saravia J, et al. PFT. 2011;8:11. Neonatal rats

EPFR Exposure Alters Neonatal Lung Architecture

Air

DCB230

Balakrishna S, Saravia J, et al. PFT. 2011;8:11. Neonatal rats

EPFRs Increase Smooth Muscle Mass in the Peribronchial Region

Balakrishna S, Saravia J, et al. PFT. 2011;8:11. Neonatal rats

EPFR Response Unique to Neonates

• Epithelial disorganization

• Increased smooth muscle mass and airway collagen deposition

• Correlated to increased airway hyper-responsiveness

EFPR Neonate

EPFR Adult

Air Neonate

E-cadherin Epithelium

α-S

MA

Sm

oo

th M

usc

le

Thevenot, et al. AJRCMB. In revision 2012. Mice

6hr Exposure to DCB230 (20 µm/cm2)

In Vitro Exposure to EPFRs Suggests EMT

Thevenot, et al. AJRCMB. In revision 2012.

In Vitro Exposure to DCB230 Alters Epithelial Cell Morphology

Vehicle DCB230 DCB230 +

Uptake Blockers

F-actin, lysosomes, nuclei

Wortmann: macropinocytosis Chlorpromazine: clathrin dependent endocytosis Filipin III: calveole, cholesterol

Thevenot, et al. AJRCMB. In revision 2012.

Gene Symbol Fold Change

E-cadherin CDH1 -1.5

Desmoplakin DSP -1.8

Junctional Adhesion Molecule F11R -1.4

Vimentin VIM 1.3

Fibronectin 1 FN1 1.3

Occludin OCLN -1.2

Snail SNAI1 1.8

Slug SNAI2 2.1

Smuc SNAI3 3.9

Zinc Finger E-box Binding Homeobox 1 ZEB1 1.5

Zinc Finger E-box Binding Homeobox 2 ZEB2 1.6

Versican VCAN 3

Matrix metalloproteinase 3 MMP3 40

Matrix metalloproteinase 9 MMP9 3

Plasminogen Activator Inhibitor SERPINE1 12.8

Frizzled 7 FZD7 1.3

Wingless Type 11 WNT11 5.9

Wingless Type 5A WNT5A 3.9

Wingless Type 5B WNT5B 5.7

β-catenin CTNNB1 -1.4

Glycogen Synthase Kinase 3β GSK-3β -1.2

Epithelial to Mesenchymal Transition Pathway Array

Ce

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ell J

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Re

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an

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Me

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Thevenot, et al. AJRCMB. In revision 2012.

Experimental Methods

Analysis

EMT

Air:Liquid Interface with Neonatal Airway Epithelial Cells

DCB-230 200µg/m3 20 min/d

EMT

Airway Remodeling

Mouse Neonate Nose Only Exposure Model

EMT Gene and

Protein

Expression

24hr post-

exposure

Protocol Time (d)

Mouse age (d)

0 7 5

11 4 9

2

6

1

5

3

7

4

8

6

10

Summary of Results

• Infant exposures to EPFR-containing PM lead to long-term pulmonary consequences – Distinct pathologies

• Inflammation • Remodeling (w/i 4d exposure) – EMT

– In vivo » E-cad + aSMA » Bgal + aSMA

– In vitro neonatal ALI » E-cad + aSMA » Expression of genes associated with EMT: ↑Snai1 + aSMA and ↓E-cad

– Respiratory dysfunction – Uptake & Oxidative stress

• 8-isoprostanes • GSH:GSSG ratio

• Relevance: – Mechanistically link PM exposure to airway remodeling – Loss of epithelial integrity suggests a window of vulnerability to RTI

Balakrishna S, et al. PFT. 2011;8:11. Wang P, et al. AJRCMB. 2011. 45: 977-983

Impact

• Global Environmental Health – Role of inhalation exposures in the predisposition, development,

and/or exacerbation of respiratory diseases

– Mechanisms by which EPFR-containing PM • induces pulmonary inflammatory diseases

• Increases susceptibility to LRTI

– Potential therapeutic targets for preventing EPFR & PM induced pulmonary dysfunction

• Environmental Policy – Enhance monitoring practices to include EPFRs

– Alter air quality standards for EPFR containing PM

Fine spheres (red spheres, 1.6 mm, yellow arrow head) Ultrafine spheres (green spheres, 0.2 mm, white arrow)

Distribution of fluorescent microspheres in the lung

Acknowledgements • Cormier Lab (LSUHSC – NO)

– Postdoctoral Fellows • Paul Thevenot, PhD • Pingli Wang MD, PhD • Dahui You, PhD

– Graduate student • Jordy Saravia

– Undergraduate students • Joseph Giaimo

– Former Students/Postdocs • Shrilatha Balakrishna, PhD • Baher Fahmy, PhD

• Barry Dellinger (LSU – BR) – Slawo Lomnicki

• Tammy Dugas (LSUHSC – S)

• Funding NIEHS: RO1 ES015050

NIEHS: P42ES013648

The project described was supported by Grants from the National Institute of Environmental Health Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute Of Environmental Health Sciences or the National Institutes of Health.